This invention relates to antibody purification by affinity chromatography techniques and, more particularly, to antibody-specific solid phase immunoadsorbents for use therein.
Immune serum globulin (ISG), also referred to as gamma globulin, is a fraction of blood plasma which is rich in antibodies, and is commercially prepared from large pools of plasma by conventional fractionation techniques well known in the art. Many clinically important antibodies are present in immune serum globulin in varying concentrations. With respect to certain diseases, such as measles and infectious (type A) hepatitis, the antibodies thereto are present in the immune serum globulin in sufficiently high concentrations so that the immune serum globulin as such is an effective prophylactic agent against these diseases. While other antibodies, such as antibody to serum (Type B) hepatitis, rubella and varicella-zoster viruses, are present in immune serum globulin, their concentration is generally too low for immune serum globulin to be prophylactically effective against these diseases. Moreover, normal immune serum globulin has generally not been regarded as an economically practical source for obtaining these low concentration antibodies in more purified form.
For prophylaxis against serum hepatitis and other diseases against which immune serum globulin is prophylactically ineffective, the current practice has been to prepare specific immune globulin by fractionating the blood of persons who have a large amount of the required antibody. Since such persons are not common, a large number of expensive screening tests must be performed to select the best donors. In the case of hepatitis B antibody, for example, only about 4% of blood donors have detectible levels, and tests may cost up to $1.50 per donor.
Among the various fractionation procedures which have previously been proposed for purification of specific antibodies is affinity chromatography, employing a solid phase immunoadsorbent comprising the antigen specific to the specific antibody bound to an insoluble carrier material. This technique involves the removal of the specific antibody from the starting solution thereof by selective complexing of the specific antibody with the antigen moiety of the immunoadsorbent, and subsequent elution of the specific antibody from the immunoadsorbent by dissociation of the antibody-antigen complex. While antibody purification by affinity chromatography has previously been employed in relatively small-scale operations with regard to antibodies primarily of laboratory interest, this technique has generally not been regarded as an economically practical means for relatively large-scale commerical preparation of clinically significant specific immune globulin, primarily due to the expense and difficulties involved in the preparation of suitable immunoadsorbents.
In order to be practical for use in large-scale antibody purification, an immunoadsorbent should meet certain basic requirements. First of all, it must be stable under the conditions required for antibody-antigen complexing and dissociation, so that under such conditions the antigen moiety of the immunoadsorbent will not be released from the carrier surface. Secondly, it should have a high complexing specificity for the specific antibody being purified, so as to minimize the amount of nonspecific protein carried over into the purified product. Thirdly, it should have a high complexing efficiency for the specific antibody, i.e., it should be capable of complexing a high percentage of the total specific antibody applied thereto. Fourthly, it should have a reasonably high complexing capacity for the specific antibody, i.e., it should be capable of complexing a reasonably high amount of specific antibody per unit weight of immuno-adsorbent. Additionally, it should be capable of providing a reasonably high yield of the specific antibody, i.e., the percentage of the complexed antibody that is released from the immunoadsorbent during elution, since this determines the number of complexing sites available on the immuno-adsorbent for reuse and hence the life span of the immuno-adsorbent.
The major obstacles to economically preparing immunoadsorbents meeting the above criteria have been the difficulties and expense involved in effectively, selectively and stably binding to the carrier surface sufficient amounts of the particular antigen specific to the specific antibody to be purified. The surfaces of the carrier materials typically employed for this purpose generally do not possess the requisite combination of high binding affinity, selectivity and capacity for the particular antigen to be bound. For this reason, immunoadsorbent preparation has generally required the rather expensive and time-consuming procedure of first chemically reacting the carrier surface with an intermediate coupling agent to increase its binding affinity for the antigen, purifying the antigen to compensate for the non-selectivity of the carrier surface and thereby reduce nonspecific binding, and thereafter chemcially coupling the purified antigen to the carrier surface through the intermediate coupling agent.
The insoluble carrier materials which have previously been employed for preparing immunoadsorbents include both organic polymeric materials and various inorganic materials, such as glass, which is particularly advantageous since it is dimensionally stable and can be thoroughly cleaned to remove contaminants, for example, by sterilization. The use of porous glass as a carrier material is disclosed, for example, in the Weetall U.S. Pat. No. 3,652,761, issued Mar. 28, 1972, but no particular significance is attached to the particular pore size of the porous glass. Furthermore, Weetall's method of preparing immunoadsorbents requires the use of an intermediate silane coupling agent to effect adequate bonding between the surface of the glass carrier and the particular antigens contemplated. Moreover, while it is known that certain biological materials, particularly those containing lipids, will adhere to normal glass surfaces under acidic conditions and will be released therefrom under strongly basic conditions, and that it is possible by techniques based on this principle to achieve at least some degree of purification of certain lipid-containing viral antigens, such as hepatitis B surface antigen, this principle does not appear to have been previously applied to the preparation of solid phase affinity chromatography immunoadsorbents which could successfully be used to achieve significant degrees of antibody purification.